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Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells

a glycoconjugate and alkyl sugar analog technology, applied in the field of metabolic oligosaccharide engineering, can solve the problems of complex glycan structure identification and molecular level delineation of their mode of action, complex glycan structure, and low glycan complexity, so as to improve the effect of toxicity and the improvement of the method

Active Publication Date: 2008-10-30
ACAD SINIC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0065]wherein the tagged glycoconjugate includes a glycan portion; a conjugate portion; and an alkynyl functional group; and reacting the tagged glycoconjugate with a probe to produce a labeled, tagged glycoconjugate; wherein the resultant toxicity of the method is improved by at least 10% as compared to presenting an azido-derivatized sugar to produce the tagged glycoconjugate.

Problems solved by technology

Although known to be involved in physiological and pathophysiological events, the identification of many glycan structures and delineation of their mode of action at the molecular level has been complicated by their underpinning complexity.
Glycan complexity results from many factors.
They are synthesized in a non-templated, post-translational process, which means that sites of glycoconjugate glycosylation and structures within them have proven, thus far, to be minimally predictable.
This also means that glycans cannot be genetically manipulated in a similar fashion to DNA and proteins.
Heterogeneity and multivalency complicate structure-function studies and the isolation of homogenous glycans in meaningful amounts from natural sources is nearly impossible.
For the procurement of homogenous glycoconjugates / glycans synthesis is the only viable route, but remains one of the most formidable challenges in glycobiology.
The link between glycan activity and complexity has presented major challenges to deciphering their activities on an individual protein, let alone, proteomic scale.
Prior glycoprotein probes have limited utility due to issues of cellular toxicity.
However, cellular glycans are complex, heterogeneous populations, resulting from a non-template-driven process that cannot be manipulated genetically.
This complexity makes the isolation and identification of glycans for structural analysis one of the most challenging and defining tasks in glycobiology.
Currently available glycoconjugate probes can be of limited utility due to potential cellular toxicity.

Method used

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  • Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells
  • Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells
  • Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

Synthesis of 1,2,3,4-tetraacetyl alkynyl fucose (Fuc) (1, mixture of anomers; Scheme 2)

[0302]To a flask containing compound 8 (0.05 g, 0.2 mmol), TFA solution (1 ml, 90% TFA in H2O) was slowly added at 0° C. The reaction was stirred on ice for 1 h and concentrated in vacuo. The resulting residue was treated with pyridine (1 ml), N,Ndimethylaminopyridine (2.0 mg), and acetic anhydride (1 ml), stirred overnight, concentrated, and diluted with dichloromethane. This solution was then sequentially washed with 1 N aqueous HCl, saturated aqueous NaHCO3, and brine. The organic phase was dried over anhydrous Na2CO3 and concentrated. Silica gel chromatography gave product 1 (0.055 g, 80%, □-pyranoside:□-pyranoside: □-furanoside:□-furanisude=30:51:11:8) as a colorless gum (FIG. 7). Partial 1H-NMR of mixture (500 MHz, CDCl3) □ 5.74 (d, J=8.4 Hz, H-1 (□pyr)), 6.24 (s, H-1 (□fur)), 6.36 (d, J=4.8 Hz, H-1 (□fur)), 6.43 (d, J=2.6 Hz, H-1 (□pyr)); ESI-TOF-HRMS m / e calculated for (M+Na)+ C15H18O9Na 3...

example 2

Synthesis of N-4-pentynoylmannosamine (10, mixture of anomers; Scheme 3)

[0303]A mixture of D-mannosamine hydrochloride (863 mg, 4.0 mmol), N-succinimidyl 4-pentynoate 9 (781 mg, 4.0 mmol), triethylamine (1.67 ml, 12.0 mmol) in DMF (31 ml) was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo, and the residue was purified by flash column chromatography (CHCl3 / MeOH 8:1) to give N-4-Pentynoylmannosamine, 10 (898 mg, 87%); 1H-NMR (500 MHz, D2O) 2.37 (t, 2.63H, J=2.5 Hz), 2.48-2.63 (m, 10.5H), 3.38-3.42 (m, 1H), 3.52 (t, 1H, J=10 Hz), 3.63 (t, 1.63H, J=10 Hz), 3.69-3.91 (m, 7.89H), 4.05 (dd, 1.63H, J=4.5 and 10 Hz), 4.35 (dd, 1.63H, J=1.5 and 4.5 Hz), 4.47 (dd, 1H, J=1.5 and 4.5 Hz), 5.03 (d, 1H, J=1.5 Hz), 5.13 (d, 1.63H, J=1.5 Hz); 13C-NMR (125 MHz, D2O)□ 14.78, 14.91, 34.62, 34.79, 53.67, 54.50, 60.91, 60.93, 67.01, 67.28, 69.25, 70.56, 70.71, 72.47, 72.50, 76.80, 84.04, 84.45, 93.36, 93.67, 175.68, 176.41; ESI-TOF-HRMS m / e calculated for (M+H)+ C1...

example 3

Synthesis of 1,3,4,6-tetra-O-acetyl-N-4-pentynoylmannosamine (4, mixture of anomers; Scheme 3)

[0304]A mixture of 10 (123 mg, 0.500 mmol) and acetic anhydride (0.227 ml, 2.40 mmol) in pyridine (4 ml) was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo, and the residue was dissolved in CH2Cl2 and washed with water. The organic layer was dried over Na2SO4 and evaporated. The residue was purified by flash column chromatography (AcOEt / Hexane 1:4) to give 1,3,4,6-tetra-O-acetyl-N-4-pentynoylmannosamine, 4 (183 mg, 86%); 1H-NMR (500 MHz, CDCl3) □ 2.00 (s, 9H), 2.06 (s, 9H), 2.097 (s, 3H), 2.10 (s, 3H), 2.11 (s, 3H), 2.14-2.18 (m, 3H), 2.19 (s, 6H), 2.46-2.58 (m, 12H), 3.81-3.87 (m, 1H), 4.00-4.15 (m, 5H), 4.23-4.30 (m, 3H), 4.69 (dd, 2H, J=4.5 and 10 Hz), 4.82 (dd, 1H, J=4.5 and 10 Hz), 5.09 (dd, 1H, J=4.5 and 10 Hz), 5.17 (t, 1H, J=10 Hz), 5.23 (t, 2H, J=10 Hz), 5.33 (dd, 2H, J=4.5 and 10 Hz), 5.90 (s, 1H), 6.03 (s, 2H), 6.36 (d, 1H, J=9.5 Hz), 6.54 (...

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Abstract

The present disclosure relates to a method for metabolic oligosaccharide engineering that incorporates derivatized alkyne-bearing sugar analogs as “tags” into cellular glycoconjugates. The disclosed method incorporates alkynyl derivatized Fuc and alkynyl derivatized ManNAc sugars into a cellular glycoconjugate. A chemical probe comprising an azide group and a visual probe or a fluorogenic probe is used to label the alkyne-derivatized sugar-tagged glycoconjugate. In one aspect, the chemical probe binds covalently to the alkynyl group by Cu(I)-catalyzed [3+2] azide-alkyne cycloaddition and is visualized at the cell surface, intracellularly, or in a cellular extract. The labeled glycoconjugate is capable of detection by flow cytometry, SDS-PAGE, Western blot, ELISA or confocal microscopy, and mass spectrometry.

Description

RELATED APPLICATIONS[0001]This application claims priority to U.S. Provisional Patent Application Ser. No. 60 / 896,777, filed on Mar. 23, 2007, titled “Pro-alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells”, the entirety of this application hereby incorporated by reference.GOVERNMENT SUPPORT[0002]This disclosure was supported, in whole or in part, by the National Institutes of Health and the Skaggs Institute for Chemical Biology.FIELD OF THE DISCLOSURE[0003]The present disclosure provides a method for metabolic oligosaccharide engineering which uses azido and / or alkyne-bearing sugar analogs and / or precursors of fucose and sialic acid to incorporate azido and / or alkyne tags into cellular glycans that are fucosylated and sialylated. The derivatized glycan is labeled by a chemical probe comprising an azide group and a visualizable, isolatable, and / or fluorogenic group. The chemical probe binds covalently (labels) to the alkynyl and / or azido groups disp...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/50C12P19/00G01N33/00C07H5/04C07K16/00C12Q1/02
CPCG01N33/5005G01N33/5008G01N33/533G01N2400/00
Inventor WONG, CHI-HUEYHSU, TSUI-LINGHANSON, SARAH R.
Owner ACAD SINIC
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